In the initial phase of these studies, we introduced constitutive MYCN and doxycycline-inducible PAX3-FOXO1 expression constructs into immortalized human myoblasts. Treatment with doxycycline induces increased PAX3-FOXO1 expression and doxycycline removal leads to decreased PAX3-FOXO1 expression. In cell culture experiments, treatment of these engineered myoblasts with doxycycline induces oncogenic transformation in focus formation assays. In subsequent animal studies, mice were injected with these cells in an intramuscular site, and when fed a doxycycline-supplemented diet, tumors that resemble human ARMS formed within 3-4 weeks. To determine if continued PAX3-FOXO1 expression is required for tumor maintenance, doxycycline supplementation was discontinued after small tumors formed, and this discontinuation resulted in overall tumor regression associated with decreased cellular proliferation and increased myogenic differentiation and cell death. Based on these findings, this inducible system is proposed to be a model of targeted therapy against the fusion protein. In the animal studies, tumors generally recurred several weeks after doxycycline discontinuation. In most of these recurrent tumors, the fusion protein was not detectable, suggesting that these tumors are recurring by a fusion protein-independent mechanism. In support of this mechanism, recurrent tumor-derived cell lines did not express the fusion protein when grown without doxycycline, yet were transformed in culture and tumorigenic in vivo under these conditions. In addition, though primary tumor-derived lines differentiated when doxycycline was removed, recurrent tumor-derived lines proliferated continuously and did not differentiate after doxycycline withdrawal. Based on these findings, we propose that rare cells with additional oncogenic events were selected during recurrence, and the action of these new oncogenic events allow the tumor cells to become independent of the fusion protein. These oncogenic events thus provide a mechanism for resistance to the targeted therapy directed against the fusion protein. To further examine the oncogenic requirements of this model system, we used the CRISPR/Cas9 system to knock down PAX3-FOXO1 or MYCN in these engineered myoblasts. Following transduction of a CRISPR construct targeting PAX3 exon 5, expression of PAX3-FOXO1 protein was knocked down to nearly undetectable levels in the presence of doxycycline. Similarly expression of MYCN protein was knocked down to nearly undetectable levels with a CRISPR construct targeting MYCN exon 2. In oncogenicity assays, parental or primary tumor-derived cells transduced with the PAX3-specific or MYCN-specific CRISPR construct failed to form foci when treated with doxycycline, whereas control transduced cells formed numerous foci as expected. Therefore, these cells are dependent on both PAX3-FOXO1 and MYCN for oncogenicity. Similar studies were also performed in recurrent tumor-derived cells. Though PAX3-FOXO1 is not expressed in these lines in the absence of doxycycline, addition of doxycycline still can induce PAX3-FOXO1 expression, and this inducible expression can be eliminated following transduction of the CRISPR construct targeting PAX3 exon 5. In contrast to the parental line described above, introduction of the PAX3-specific CRISPR did not affect the ability of several recurrent tumor-derived lines to form foci in the absence or presence of doxycycline. These findings confirm that these recurrent tumor-derived cells do not require fusion protein expression for oncogenic transformation. To dissect the mechanistic features of the fusion-independent recurrence mechanism, we examined whether the in vivo environment is required. We divided the recurrence process into two steps - generation of fusion-independent cells and formation of a fusion-independent oncogenic lesion. We first assessed whether the in vivo environment is required for fusion-independent cells to arise. The original engineered myoblasts were grown in culture for several weeks in the presence of doxycycline, and then injected into mice fed a diet without doxycycline supplementation. In culture conditions in which PAX3-FOXO1 is highly induced, the population of cells grown in doxycycline often gave rise to doxycycline-independent tumors. Of note, the high level of PAX3-FOXO1 expression was initially associated with significant growth suppression, and the continued growth in doxycycline is postulated to select for cells that are more tolerant of the high PAX3-FOXO1 level and to enrich for cells that have PAX3-FOXO1-independent oncogenic mechanisms. This first set of studies thus indicates that the recurrence process can be recapitulated with an initial in vitro PAX3-FOXO1-dependent step followed by in vivo PAX3-FOXO1-independent conditions. In a second set of studies, we investigated whether the development of the PAX3-FOXO1-independent lesions can occur in culture. Therefore, after the cells were grown in culture with doxycycline for several weeks, the cells were plated without doxycycline in a focus formation assay. We detected a low frequency of focus formation under these conditions, and subsequent expansion of these rare foci into cell lines confirmed that these lines are transformed in the absence of doxycycline-induced PAX3-FOXO1 expression. These studies thus demonstrate that rare PAX3-FOXO1-independent cells can arise in vitro and then can form an oncogenic lesion (i.e., transformed focus) in vitro. Therefore, the in vivo environment does not appear to be required for the generation or oncogenic growth of these recurrent cells.